I have been trying to develop ELISA assay using 1536 microplate (colorimetric based assay - with HRP conjugated antibodies and TMB). however, I have encountered several difficulties, the main problem is that i am not getting any signal.
I already developed many ELISA assays (of various types) using 384 well plate. However, assays that are performing well in 384 well plate have failed to provide similar result with 1536 well plate. I have tested several protocols (different reagents and buffers) however still having the same problem.
In addition, I couldn't find any information on line (such as articles or other sources of information) regarding ELISA assay in 1536 well plate.
I would highly appreciate your advice.
Yes you are right. There are several limitations and disadvantages with high-throughput screening (HTS) micro-plate selection in ELISA.
The expected throughput of the assay is factored and while the preferred density for screening assays might be 1,536-well, assay performance or automation may only support 384- or 96-well formats. The assessment of plate density can raise questions about the well shape and volume, which will also impact the shaking efficiency and well meniscus that can be critical components in certain detection systems.
Although, microplates for HTS applications are available with various numbers of individual wells, usually from 96 to 1,536 wells per plate, each with distinct advantages and disadvantages.
It becomes very difficult to manually handle the microplate with 1,536 wells. Thus working with higher-density plates can present new technical challenges for the assay as well as the need for instrumentation capable of dispensing compounds and reagents into higher-density formats.
The overall throughput is ultimately depend on a variety of technical factors including instrumentation (e.g., plate reader speed, with well-by-well reads slower than whole-plate reads).
Yes, there are very few information available online. Hope the given link may be useful for your concern to some extent:
Microplate Selection and Recommended Practices in High-throughput Screening and Quantitative Biology.
Markossian S, Grossman A, Brimacombe K, et al., editors. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.
https://www.ncbi.nlm.nih.gov/books/NBK558077/
Thank you. The assay is fully automated. I have tested both Direct and competitive ELISA in 1536 (colorimetric assay 450nm). This is not a HT screening assay but a protein-protein interaction test. I am trying to figure out what might be the problem that may cause "no signal" result. I have already tested several reagent/antibodies concentrations and additional troubleshooting.
Hi, I do not have experience with high throughput system but I also work with protein protein interactions. TMB is light sensitive so may be try to take TMB in different intervals than the whole amount for the reaction.
You need to troubleshoot your setup, with positive controls for each step. Does the coating work? Try e.g. serial dilutions of your reporter enzyme.
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